1. Field of the Invention
This invention relates to producing virus-free biological mixtures such that valuble biological components, e.g., cells and proteins, retain their structure and function. More especially, this invention relates to the inactivation of viruses, e.g., hepatitis viruses, human immunodeficiency viurus (HIV), or other virues in human blood, blood cellular components, blood plasma and blood plasma fractions such that each remains suitable for therapeutic use. In particular, this invention relates to producing cellular blood products (e.g., whole blood, red cell concentrates, platelet concentrates and leukoiyte concentrates) and non cellular blood products (e.g., whole plasma, antihemophilic factor immune globulin, fibrinogen) which are rendered substantially free of infectious hepatitis B virus, non-A, non-B hepatitis virus, or other viral infectivity by treatment with aryl diol epoxides.
2. Background Information
Transmission of viral diseases (e.g., hepatitis B, acquired immunodeficiency syndrome, cytomegalovirus infections) through blood transfusion is a significant problem in transfusion medicine. While donor selection criteria and screening of donor blood for viral markers helps reduce the transmission of viruses to recipients, screening methods are incomplete, and it is desirable to inactivate viruses contained in donor blood without altering the structure and function of its valuable consitituents, e.g., red blood cells, platelets, leukocytes, and plasma proteins. Similarly, other biological mixtures, e.g., hybridoma cell lines, milk and sperm, can contain infections virus and it would be advantageous to inactivate said virus(es) while retaining the valuable consitutents of these mixtures.
Methods typically used for the inactivation viruses, such as those useful in the preparation of viral vaccines, generally destroy the function and structure of cells and proteins. For instance, in the preparation of a hepatitis B virus vaccine, it is common practice to heat the prearation at temperatures in excess of 80.degree. C. and to treat with formaldehyde. These treatments not only inactivate viral infectivity, but also irreversibly damage blood cells and proteins, rendering them unsuitable for transfusion.
Recently, both physical and chemical methods have been developed which inactivate viruses contained in blood, while retaining blood protein structure and function. Protein solutions heated either following lyophilization or in solution in the presence of high concentrates of sugars and/or amino acids have been shown to have greatly reduced viral infectivity, while retaining functional activity of many proteins. Chemical methods in use for the preparation of protein mixtures include beta-propiolactone and the use of solvent/detergent mixtures, especially tri(n-butyl)-phosphate. However, application of these methods to cells generally results in disruption and inactivation.
As a result of the foregoing, viral inactivation methods are not commonly applied to the preparation of whole blood and blood cell components derived therefrom. Rather, viral safety relies solely on donor selection and donor blood screening; methods known to be useful, but insufficient. Thus, the recipients of these products must accept the risk that they may be contaminated with hepatitis viruses, HIV, cytomegalovirus or other infectious viruses. As a result, these recipients may suffer liver damage or damage to other organ systems, illness, incapacitation, and occassionally death.
Other methods for inactivation of viruses in cellular products include ultraviolet light, gamma-irradiation, or the use of beta-propiolactone. Each method can be characterized as being non-specific, modifying nucleic acid, other cell structures and proteins alike. Thus, for example, the use of ultraviolet light has been shown to inactivate viruses in a platelet concentrate, however, severe platelet damage resulted from higher intensitites. Beta-propiolactone reacts with nucleic acid and protein with similar rate constants; thus, while viruses can be activated, more than half of the factor VIII content of plasma is lost.
Yet another problem is that some of the viruses contaminating blood or other biological fluids are contained within the cell, either as a fully formed virus or in the form of free viral nucleic acid integrated into the host genome. For instance, the human immunodeficiency virus is contained within leukocytes. It is a special concern to be able to inactivate both cell-free and cell-contained forms of virus, while retaining the structure and function of cells not containing virus. Fortunately, not all viruses are contained within the cells of interest and the functionality of some cells, e.g., red blood cells or platelets, do not require cell division following transfusion.
It is to be understood that the problems of inactivation of the viruses in valuable biological mixtures are distinct from the problems of inactivation of the viruses themselves due to the copresence of the desirable proteinaceous components of the plasma. Thus, while it is known how to inactivate the hepatitis B virus by using crosslinking agents, for example, glutaraldehyde, nucleic acid reacting chemicals, for example, formaldehyde, or oxidizing agents, for example chlorox, etc., it has been believed that these methods are not suitable for the inactivation of the virus in blood due to the observation that most of these activating agents, e.g., glutaraldehyde, sodium hypochlorite or formaldehyde, denatured the valuable proteinaceous components of the plasma.
Problems may also exist in deriving valuable proteins from non-blood sources. These sources include, but are not limited to, mammalian milk, ascitic fluid, serum, saliva, placental extracts, tissue culture cell lines and their extracts, including transformed cells, and products of fermentation. For instance, the human lymphoblastoid cells have been isolated which produce alpha-interferon. However, the cell line in commercial use today contains Epstein-Barr virus genes. It has been a major concern that the use of interferon produced by these cells would transmit viral infection or induce viral caused cancerous growth.
The present application concerns the action of aryl diol epoxides to inactive viruses and simultaneously to retain labile protein activity.
Aryl diol epoxides are known to form adducts with DNA in vitro and in vivo (H. B. Gamper et al, (1980), Proc. Nat. Acad. Sci. (USA), 77:2000). Several studies have indicated anti-viral effects of these agents (M. L. Lockhart et al, (1986), Chemico-Biological Interact., 58: 217; G. T. Chang et al, (1981), Biochem. Biophys Res. Comm., 100:1337; and G. T. Bowden et al, (1986), Chemico-Biological Interact., 58:333). However, heretofore there was no indication that these compounds have been used as antiviral agents specifically in the context of the sterilization of cellular components of blood.
The present invention is directed to achieving three goals, namely, (1) a safe, (2) viral inactivated protein-containing composition, (3) without incurring substantial protein denaturation. As shown above, these three goals are not necessarily compatible since, for example, glutaraldehyde inactivates viral infectivity, but fixes cells and substances such as beta-propiolactone inactivate viruses, but also substantially denaturate valuable plasma proteins, for example, factor VIII.
It, therefore, became desirable to provide a virus inactivation process for obtaining cell- or protein-containing compositions which does not substantially inactivate cells or denature the valuable protein components therein. More especially, it is desirable to provide blood cell and blood protein-containing compositions in which substantially all of the hepatitis viruses and other viruses present are inactivated, but which retains at least 60% and preferably 80% intact cells, (e.g., red blood cells) and active protein (e.g., immunoglobulin).
It is a further object of the present invention to provide products from other biological fluids, from cancer or normal cells or from fermentation processes following gene insertion which are substantially free of infectious virus.